To determine if mutations in the nucleotide binding domain of NOD2 leads to dysregulation of TLR responses we assessed the severity of TNBS-colitis in mice that over-express NOD2 bearing a Blau mutation (R314W), NOD2 bearing a Crohn's disease frame-shift mutation (FS987) or intact NOD2. Over-expression was achieved either by administration of expression plasmids encapsulated in HVJ-E (Sendai virus envelope) or by the presence of a transgene. We found that whereas over-expression of intact NOD2 protected mice from the development of TNBS-colitis, NOD2 with either a Blau or Crohn's mutation did not. These studies were repeated in mice bearing a Blau NOD2 transgene who lack normal NOD2 and again it was observed in this case as well that Blau NOD2 does not exert regulatory function. In further in vitro studies of bone marrow dendritic cells from transgenic and control mice we found that the mice bearing a Blau NOD2 transgene exhibited greatly reduced NF-kappaB and MAPK responses and produced greatly reduced levels of pro-inflammatory cytokines in spite of the fact that they still expressed normal NOD2 genes. This plus the results of the TNBS-colitis studies mentioned above indicates that the abnormal Blau NOD2 exerts a dominant negative effect on the normal NOD2. In addition, these data indicate that Blau NOD2 function is defective rather than hyper-active as previous thought. In further studies we conducted studies of responses of patients with Blau syndrome and found that as in the case of transgenic mice, the patient dendritic cells exhibited reduced TLR responses. Finally, we conducted extensive Western blot studies to elucidate the signaling pathway elicited by NOD2 ligand in mice and humans with Blau mutations. We found that Blau NOD2 does not undergo normal dimerization and leads to the activation of an unstable RICK adaptor protein as well as a poor IRF4 signal, the latter a previously identified TLR inhibit activated by NOD2. Similar findings were observed in Western blot studies of dendritic cells from Blau patients. It is likely that the disturbance in IRF-4 activation underlies the dominant-negative effect of the Blau mutation.